Development of multiplex RT‐ddPCR assays for detection of SARS‐CoV‐2 and other common respiratory virus infections

Abstract Background Measures for mitigation of Coronavirus Disease 2019 (COVID‐19) were set to reduce the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). SARS‐CoV‐2 and other respiratory viruses share similar transmission routes and some common clinical manifestations. Co‐circulation of SARS‐CoV‐2 and other common respiratory viruses is imminent. Therefore, development of multiplex assays for detecting these respiratory viruses is essential for being prepared for future outbreaks of respiratory viruses. Methods A panel of three reverse transcription droplet digital PCR (RT‐ddPCR) assays were developed to detect 15 different human respiratory viruses. Evaluations of its performance were demonstrated. A total of 100 local and 98 imported COVID‐19 cases in Hong Kong were screened for co‐infection with other common respiratory viruses. Results All detected viral targets showed distinct signal clusters using the multiplex RT‐ddPCR assays. These assays have a broad range of linearity and good intra‐/inter‐assay reproducibility for each target. The lower limits of quantification for all targets were ≤46 copies per reaction. Six imported cases of COVID‐19 were found to be co‐infected with other respiratory viruses, whereas no local case of co‐infection was observed. Conclusions The multiplex RT‐ddPCR assays were demonstrated to be useful for screening of respiratory virus co‐infections. The strict preventive measures applied in Hong Kong may be effective in limiting the circulation of other human respiratory viruses. The multiplex assays developed in this study can achieve a robust detection method for clinical and research purposes.


| INTRODUCTION
Hong Kong and Mainland China follow the 'dynamic-zero' strategy to control Coronavirus Disease 2019 . 1 The practice of social distancing, mandatory mask-wearing and extensive screening of travellers/local people has been used to reduce Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission in Hong Kong.
SARS-CoV-2, like other respiratory viruses, is transmitted mainly via respiratory route in close proximity of or direct contact with asymptomatic or symptomatic individuals.
There is increasing evidence supporting the idea that SARS-CoV-2 is going to be an endemic disease in humans and hence cocirculation with other common respiratory viruses in nature becomes inevitable. 2 Previous studies have reported that the detection rate of other respiratory viruses among all SARS-CoV-2 positive cases was about 3.4% to 7.3%. 3,4 Such respiratory virus co-infections may aggravate the severity of clinical manifestations and increase the rate of both those needing intensive care units and deaths of in-patients. 5 The prevalence of viral co-infections is yet to receive enough attention. There is a need of prompt identification of patients' co-infected with different respiratory viruses, thereby allowing timely treatments for this high-risk group. Besides, there is also a need of monitoring the virus co-circulation patterns in community for informing public health policy actions. Here, we report development of multiplex reverse tran-

| Primer and probe sequences
Three assays with primer/probe sets targeting 15 common respiratory virus genes (Table S1) were developed: (1) Assay 1 detects influenza A virus M gene (Set 1a), 6 PIV1 HN gene (Set 1b), 7 SARS-CoV-2 RdRp gene (Set 1c), 8 Adv hexon gene (Set 1d) 9 and RSV N gene (Set 1e) 10 ; (2) Assay 2 detects influenza B virus M gene (Set 2a), 6 PIV2 HN gene (Set 2b), 10 PIV3 HN gene (Set 2c), 10 PIV4 nucleocapsid gene (Set 2d), 11 EV/RV 5 0 -UTR gene (Set 2e) 12 and HMPV N gene (Set 2f) 13 ; and (3) Assay 3 detects HCoV-229E N gene (Set 3a), 14 HCoV-NL63 N gene (Set 3b), 14 HCoV-HKU1 N gene (Set 3c) 14 and HCoV-OC43 N gene (Set 3d). 14 Several primer/probe sets (Sets 1d, 1e, 2b, 2c, 2e and 2f) were modified by reducing primer mismatches to our up-to-date virus sequence datasets, whilst others were obtained from previously published work as shown. The modified primer/probe sets were analysed in silico by an in-house R program using the complete human virus sequences from Virus Pathogen Resources (viprbrc.org/), Influenza Research Database (fludb.org/) and National Centre for Biotechnology Information (ncbi.nlm.nih.gov/). The modified primers were considered optimal when they fulfilled all of the following criteria: (1) the last five bases from 3 0 end perfectly match with their target sequences; (2) the last 10 bases from 3 0 end do not have more than one mismatch to their targets and (3) the total number of mismatches does not exceed three bases. No modified probe should have more than one mismatch with the target sequences. All primers and probes were synthesized by Integrated DNA technologies with the label of 5 0 -fluorophore (FAM or HEX), 3 0 -Iowa Black FQ quencher and an internal ZEN quencher. Viral RNA of these samples was extracted using QIAamp viral RNA mini kit (Qiagen) and the extracted RNA samples were stored at À80 C before further analyses.

| The RT-ddPCR assays for respiratory viruses
A total of 20 μl of standard PCR reaction mixture for all three assays was prepared by one-step RT-ddPCR Advanced Kit for Probes

| Preparation of plasmid standards
For evaluation of the respiratory virus panel, plasmid standards were prepared using RT-ddPCR products and Zero Blunt ® TOPO ® PCR Cloning Kits (Invitrogen) based on manufacturer's instructions. The plasmid standards were confirmed by Sanger sequencing. All plasmids were digested by EcoRI (Thermo Scientific) following the manufacturer's instructions before subsequent experiments.

| Evaluation of the respiratory virus panel
The cross-reactivity and the limit of blank (LoB) was analysed by testing the mixture of the plasmid standards (10 4 copies each, without the testing assay's targets) spiked in RNA extracted from negative human respiratory samples. The LoB for each assay was determined as 95th percentile of positive droplets for FAM and HEX signals in 20 replicates of negative reaction. Fifteen plasmid standards (Adv, HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-OC43, HMPV, PIV1, PIV2, PIV3, PIV4, RSV, RV, SARS-CoV-2, H1 and Vic) were chosen to test the performance of the assays. Tenfold serial dilutions of the plasmid standards ranging from 10 5 to 10 0 were done to determine the dynamic range, intra-assay and inter-assay reproducibility. The intraassay reproducibility was determined by testing different concentrations in triplicates from the same run. The inter-assay reproducibility was determined by testing two more replicates in another two different days within a week. Then, the dynamic range was determined by these five replicates from three different runs. The limit of quantification (LoQ) was determined by the lowest concentration of the plasmid standard of each target to be quantified in 16 replicates with CV ≤ 25%. 15 The LoQ analyses were run for three different days (triplicates on day 1, five replicates on day 2 and eight replicates on day 3). The data generated for RT-ddPCR performance were analysed using Prism 9.

| Assessment and evaluation of RT-ddPCR assays for respiratory virus detections
Based on the designed criteria, the modified primer/probe sets have a good match to the great majority of our studied sequence targets (95.17% to 100%; Table S1). All archived positive control samples tested positive in the corresponding RT-ddPCR and distinct positive signal clusters were observed in these reactions (Figures 1-3, blue or green droplets).  All three assays were tested by human respiratory samples that were negative for the studied viruses. No cross-reactivity was observed for any primer/probe sets, demonstrating that these assays were highly specific. Based on the results of cross-reactivity study, the number of false positive droplets in negative reactions was determined. The LoB in FAM channel of Assays 1-3 was 4, 7 and 9 droplets, respectively. The LoB in HEX channel of Assays 1-3 was 5, 8 and 6 droplets, respectively. For each assay, multiple replicates were tested for intra-assay (N = 3) and inter-assay reproducibility (N = 5) (Table S2 for Assay 1, Table S3 for Assay 2 and Table S4 for Assay 3).
All assays had a CV ≤ 25%, with lower concentration of standards tending to have a higher CV value of variability as expected. At least four orders of magnitude in dynamic range were displayed for PIV4  (Tables S2-S4), meeting the recommended standard for microbial detection. 15 LoQ values of targets in copies per reaction were no more than 33 for Assay 1, 46 for Assay 2 and 45 for Assay 3.

| Co-infections with SARS-CoV-2 and other common respiratory viruses
The established assays were used to test 198 SARS-CoV-2 positive human specimen for respiratory virus co-infection (Table 1) 21 Previous study showed that co-infections of Adv or RSV with SARS-CoV-2 had no differences in in-patient outcomes compared with SARS-CoV-2 mono-infections. 22 Analysing serial samples of co-